增强核酸疫苗免疫效果的策略及其作用机制的研究

发布时间：2018-03-09 04:09

本文选题：核酸疫苗　切入点：乙肝治疗性DNA疫苗　出处：《第二军医大学》2006年博士论文　论文类型：学位论文

【摘要】：核酸疫苗(DNA疫苗)是指将含有编码抗原基因的真核表达载体直接接种体内,在体内表达相应抗原刺激机体产生针对该抗原的免疫应答,产生保护性免疫。核酸疫苗由于其独特的双免疫激活作用而受到越来越多的关注,有一些核酸疫苗已经进而Ⅱ期、Ⅲ期临床研究,然而,核酸疫苗的免疫效果却始终不甚理想,在动物实验中,对于猴子、猩猩等大动物,疫苗用量过大,为此,探索增强核酸疫苗免疫效果及降低疫苗用量的策略研究是十分必要的。剂型改造是提高DNA疫苗免疫效果的方法之一。本课题中探讨了一种新型的DNA疫苗给药系统,用聚乳酸/羟基乙酸共聚物(PLGA)包裹DNA疫苗形成微球疫苗后免疫小鼠,观察免疫后小鼠体液免疫、细胞免疫及粘膜免疫的效果。实验中,以乙肝治疗性DNA疫苗和多表位口蹄疫DNA疫苗为候选疫苗,应用PLGA包裹形成微球疫苗,首先在体外实验中验证了疫苗微球对抗原递呈细胞的靶向作用。小鼠巨噬细胞系Raw264.7细胞被用于体外吞噬实验,模拟抗原递呈细胞对疫苗抗原的摄取过程。通过姬姆萨染色,证实了微球疫苗的可吞噬性;荧光标记的疫苗微球的应用,可以更准确的动态观察微球的吞噬过程,在吞噬的2小时、4小时、8小时后分别收集细胞荧光显微镜观察,可以看到随着时间延长,细胞内点状荧光逐渐融合,荧光强度逐渐增加,进一步证实微球的可吞噬性。吞噬后的微球能否成功释放疫苗,疫苗抗原能否正确表达?后续实验中,在体内、体外两个层面上证实了抗原成份在抗原递呈细胞中的正确表达。当应用PLGA包裹的乙肝DNA疫苗口服免疫小鼠后,可全面的激发机体的细胞免疫和体液免疫,还可在局部引流淋巴结中观察到疫苗抗原的表达,观察到体现粘膜免疫效果的IgA的分泌,充分证实了口服乙肝DNA疫苗良好的免疫效果,获得了有应用前景的口服治疗性乙肝DNA疫苗。在多表位口蹄疫DNA疫苗的研究中发现,PLGA包裹可以降低疫苗的免疫剂量、减少疫苗的免疫次数,使新型口蹄疫DNA疫苗更廉价、使用更方便。膜联蛋白B1基因是本课题组以免疫筛选法从猪囊尾蚴cDNA文库中得到的一个新基因,其基因产物是猪带绦虫囊虫病的诊断用抗原,也是很好的保护性抗原,以annexin B1为抗原基础构建的囊虫DNA疫苗,在猪的保护性实验中可保护仔猪免于虫卵冲击后的感染或提高减虫率。对于一个高免疫原性的疫苗抗原,其结构的解析、生物学活性的认识是安全、有效的利用疫苗抗原的基础,为了找到能
[Abstract]:Nucleic acid vaccine (DNA vaccine) means that the eukaryotic expression vector containing the encoded antigen gene is directly inoculated into the body and the corresponding antigen is expressed in the body to stimulate the body to produce an immune response to the antigen. The nucleic acid vaccine has attracted more and more attention because of its unique double immune activation. Some nucleic acid vaccines have gone on to phase II and III clinical studies. However, the immune effect of nucleic acid vaccine has not been satisfactory. In animal experiments, the dosage of vaccine is too large for big animals such as monkeys and orangutans. Therefore, it is necessary to explore the strategy of enhancing the immune effect of nucleic acid vaccine and reducing the dosage of vaccine. Dosage form modification is one of the methods to improve the immune effect of DNA vaccine. In this paper, a new DNA vaccine delivery system was studied, in which mice were immunized with DNA vaccine encapsulated with polylactic acid / glycolic acid copolymer. To observe the effect of humoral immunity, cellular immunity and mucosal immunity of mice after immunization, the therapeutic DNA vaccine of hepatitis B and the DNA vaccine of multiepitope foot-and-mouth disease were used as candidate vaccines, and the microsphere vaccine was formed by PLGA encapsulation. The mouse macrophage cell line Raw264.7 cells were used for phagocytosis in vitro, mimicking the process of antigen uptake by antigen presenting cells. The results showed that the phagocytosis of the microsphere vaccine was confirmed, and the application of fluorescent labeled vaccine microspheres could more accurately observe the phagocytosis process of the microspheres, and the fluorescence microscope was collected after 2 hours and 4 hours and 8 hours of phagocytosis, respectively. It can be seen that as the time goes on, the intracellular dot fluorescence gradually fuses and the fluorescence intensity increases, which further proves the phagocytosis of the microspheres, and whether the phagocytic microspheres can release the vaccine successfully, and whether the vaccine antigen can be correctly expressed? In subsequent experiments, in vivo and in vitro, the correct expression of antigen components in antigen-presenting cells was confirmed. When mice were immunized orally with PLGA encapsulated hepatitis B DNA vaccine, the cellular and humoral immunity of the body could be fully stimulated. The expression of vaccine antigen was also observed in the local drainage lymph nodes, and the secretion of IgA, which reflected the mucosal immune effect, was observed, which fully confirmed the good immune effect of the oral hepatitis B DNA vaccine. In the study of multiepitope foot-and-mouth disease (FMD) DNA vaccine, we found that PLGA-encapsulated vaccine can reduce the dose of vaccine, reduce the number of times of immunization, and make the new DNA vaccine cheaper. It is more convenient to use. Annexin B1 gene is a new gene obtained from cDNA library of cysticercus cellulosae by immune screening method. Its gene product is the diagnostic antigen of Taenia solium cysticercosis, and it is also a good protective antigen. The cysticercosis DNA vaccine constructed on the basis of annexin B1 can protect piglets from infection or increase the rate of worm reduction in the protective experiment. For a vaccine antigen with high immunogenicity, its structure is analyzed. The understanding of biological activity is the basis for the safe and effective use of vaccine antigens.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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